Under the provisions of Section 119 of 35 U.S.C., Applicants hereby claim the benefit of the filing date of European Patent Application Number 98121299.6, filed Nov. 9, 1998.
This invention relates to a compound named Vancoresmycin, which is obtainable by cultivation of the microorganism HIL-006734 (DSM 12216), and to its pharmaceutically acceptable salts and derivatives. The present invention further relates to a process for the production of Vancoresmycin, to the microorganism HIL-006734 (DSM 12216), to the use of Vancoresmycin and its pharmaceutically acceptable salts and derivatives as pharmaceuticals, including their use as antibiotics, and to pharmaceutical compositions comprising Vancoresmycin or a pharmaceutically acceptable salt or derivative thereof.
Methicillin resistant Staphylococcus aureus (MRSA) infections are known to be predominant in several infectious conditions, for example, wounds and burns. Vancomycin and teicoplanin, belonging to the glycopeptide class, are the only two antibiotics clinically used for the treatment of MRSA infections. Due to the recent emergence of vancomycin- and teicoplanin-resistant strains, however, these infections are reported to have become menacing and fatal. In response, an intensive search for a structurally different class of compounds active against these vancomycin- and teicoplanin-resistant strains has been initiated. For instance, methylsulfomycin I, a cyclic thiopeptide, has been described earlier (European Patent Publication No. 0818539 filed Jul. 11, 1996) as an antibiotic active against vancomycin- and teicoplanin-resistant strains.
It has now been found that a novel compound named Vancoresmycin has antibiotic activity.
The present invention relates to Vancoresmycin, a compound of the formula: 
and to its pharmaceutically acceptable salts and derivatives, such as esters, ethers, and obvious chemical equivalents, including all stereoisomeric forms and all tautomeric forms.
Vancoresmycin has the molecular formula C71H126N2O21 (MW1343.80) and may be characterized by any one or more of its physico-chemical and spectral properties given below, such as its 1H NMR spectroscopic data and its 13C NMR spectroscopic data, both provided in Table 2.
Vancoresmycin is a new antibiotic active against vancomycin- and teicoplanin-resistant strains. It has a hitherto unreported structure with a tetramic acid moiety bearing an acyl substituent at the 3-position. This acyl substituent has a highly oxygenated long alkyl chain substituted with an amino sugar. A chemical abstract literature search established Vancoresmycin to be a new compound.
Vancoresmycin may be obtained by cultivating a microorganism referred to as culture No. HIL-006734 (henceforth referred to as HIL-006734). This microorganism was isolated from a soil sample collected from National Park, Borivli, Mumbai, India. The microorganism HIL-006734 belongs to the order of Actinomycetalos, genus Amycolatopsis. It was deposited on Jun. 4, 1998 with the German Collection of Microorganisms and Cell Cultures (DSMZxe2x80x94Deutsche Sammlung von Mikroorganismen und Zelikulturen GmbH), Braunschweig, Germany, and has been given the accession number DSM No. 12216.
The present invention further provides a process for the production of Vancoresmycin from Amycolatopsis species HIL-006734, its mutants and variants, comprising the steps of: growing the Amycolatopsis species HIL-006734 under aerobic conditions in a nutrient medium containing one or more sources of carbon and one or more sources of nitrogen and optionally nutrient inorganic salts and/or trace elements; isolating the Vancoresmycin compound; and purifying the Vancoresmycin compound in a customary manner.
Mutants and variants of the microorganism HIL-006734 may also be able to synthesize the compound according to the present invention. Such mutants may be produced in a known manner by physical means, for example irradiation such as with ultraviolet- or X-rays, or chemical mutagens, such as ethylmethylansulfonate (EMS), 2-hydroxy4-methoxy-benzophenone (MOB) or N-methyl-Nxe2x80x2-nitro-N-nitrosoguanidine (MNNG).
The screening for suitable mutants and variants which can produce the compound according to the invention can be confirmed by determination of the biological activity of the active compounds accumulated in the culture broth, for example by testing the antibacterial action.
The nutrient medium preferably contains sources of carbon, nitrogen and nutrient inorganic salts. The carbon sources are, for example, starch, glucose, sucrose, dextrin, fructose, molasses, glycerol, lactose, or galactose. A typical carbon source is starch. The sources of nitrogen are, for example, soybean meal, peanut meal, yeast extract, beef extract, peptone, malt extract, corn steep liquor, gelatin, casamion acids. Peptone and yeast extract are typical. The nutrient inorganic salts are, for example, sodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, calcium chloride, calcium carbonate, potassium nitrate, ammonium sulphate, and magnesium sulphate. Calcium carbonate, sodium chloride, and magnesium sulphate are typical.
The cultivation of HIL-006734 may be carried out at temperatures between 25-35xc2x0 C. and pH between 6.0 and 8.0. Typically, HIL-006734 is cultivated at 30xc2x0 C. (xc2x11xc2x0 C.) and pH 7.0.
A good yield of the antibiotic of the invention may be obtained by cultivating HIL-006734 for 60-96 hours. Typically, cultivation is carried out by fermentation for 68-96 hours under submerged conditions, for example in shake flasks, as well as in laboratory fermenters. The progress of fermentation and formation of the Vancoresmycin can be detected by High Pressure Liquid Chromatography (HPLC) and by measuring the bioactivity of the culture broth against Staphylococci and Enterococci species by the known microbial agar plate diffusion assay method. The preferred culture is Staphylococcus aureus 3066, which is a resistant strain to methicillin, a pxcex2lactam antibiotic reported in the literature, and Entrococcus faecium (E. faecium VR-1), which is resistant to vancomycin. In the resulting culture broth, Vancoresmycin is present in the culture filtrate as well as in mycelium and can be isolated using known separation techniques. Thus, it can be recovered from the culture filtrate by extraction at pH 5-8 with a water immiscible solvent such as ethyl acetate, dichloromethane, chloroform, or butanol, or by hydrophobic interaction chromatography using polymeric resins such as xe2x80x9cDiaion HP-20(copyright)xe2x80x9d (Mitsubishi Chemical Industries Limited, Japan), xe2x80x9cAmberlite XAD(copyright)xe2x80x9d (Rohm and Hass Industries U.S.A.) activated charcoal, or by ion exchange chromatography at pH 5-8. Typically, the active material is extracted with ethyl acetate. The active material can also be recovered from mycelium by extraction at pH 5-8 with a water miscible solvent such as methanol, acetone, acetonitrile, n-propanol, or iso-propanol. Alternatively, it may be extracted at pH 5-8 with a water immiscible solvent such as ethyl acetate, dichloromethane, chloroform, or butanol. Typically, the active material is extracted at pH 5-8 with ethyl acetate. Concentration and lyophilization of the extracts gives the active crude material.
The antibiotic Vancoresmycin of the present invention may, for example, be recovered from the crude material as follows:
By fractionation using any of the following techniques: normal phase chromatography (using alumina or silica gel as stationary phase; eluents such as petroleum ether, ethyl acetate, methylene chloride, acetone, chloroform, methanol, or combinations thereof; and additions of amines such as NEt3); reverse phase chromatography (using reverse phase silica gel such as dimethyloctadecylsilylsilica gel, (RP-18) or dimethyloctylsilyl silica gel (RP-8) as stationary phase; and eluents such as water, buffers (for example, phosphate, acetate, citrate (pH 2-8)), and organic solvents (for example, methanol, is acetonitrile, acetone, tetrahydrofuran, or combinations of these solvents)); gel permeation chromatography (using resins such as (copyright)Sephadex LH-20 (Pharmacia Chemical Industries, Sweden), TSKgel (copyright)Toyopearl HW (TosoHaas, Tosoh Corporation, Japan) in solvents such as methanol, chloroform, acetone, ethyl acetate, or their combinations, or (copyright)Sephadex G-10 and G-25 in water); or by counter-current chromatography (using a biphasic eluent system made up of two or more solvents such as water, methanol, ethanol, iso-propanol, n-propanol, tetrahydrofuran, acetone, acetonitrile, methylene chloride, chloroform, ethyl acetate, petroleum ether, benzene, and toluene). These techniques may be used repeatedly, alone or in combination. A typical method is chromatography over reverse phase silica gel (RP-18).
The compound Vancoresmycin may be converted into pharmaceutically acceptable salts and derivatives, like esters and ethers, and other obvious chemical equivalents, which are all covered by the present invention. The salts and derivatives can be prepared by standard procedures known to one skilled in the art. Salts like sodium and potassium salts, for example, may be prepared by treating Vancoresmycin with suitable sodium or potassium bases.
Esters and ethers may be prepared by the methods given in the literature, for example, in Advanced Organic Synthesis, 4th Edition, J. March, John Wiley and Sons., 1992.
The amino group of the sugar moiety can be alkylated or acetylated, e.g. with acid chlorides by standard procedures known to one skilled in the art.
Chemical equivalents may be stable complexes with metal ions, e.g. transition metals like La3+, Sm3+, Eu3+, Gd 3+, which are typical for tetramic acid derivatives and may be prepared by the methods given in the literature (K. Tanaka et. al., Chem. Pharm. Bull. 1979, 27,1901; K. Matsuo, Chem. Pharm. Bull. 1980, 28, 2494).
The double bonds of the alkyl side chain may be reduced by the methods given in the literature, for example in P. N. Rylander, xe2x80x9cHydrogenation Methodsxe2x80x9d, Academic Press, New York (1985), Chpt. 2, or may be hydrohalogenated by methods described by H. O. House in xe2x80x9cModern Synthetic Reactionsxe2x80x9d, W. A. Benjymin, Inc., New York (1972), pp 446-452. Hydroxylated derivatives may be produced by reaction of the double bonds with reagents such as OsO4 as described in the literature, e.g. in Chem. Rev. 1980, 80, 187.
Derivatives may also be formed by conversion of the double bonds into epoxides by oxidation, e.g. with MCPBA, as described in Advanced Organic Synthesis, 4th Edition, J. March, John Wiley and Sons., 1992.
Vancoresmycin has antibacterial activity. Minimum inhibitory concentrations of Vancoresmycin against a wide range of bacterial are given in Table 3 below. Vancoresmycin and its pharmaceutically acceptable salts and derivatives can be administered to animals, such as mammals, including humans, as pharmaceuticals on their own, in mixtures with one another, and in the form of pharmaceutical compositions that permit parenteral administration. Accordingly, the present invention also relates to Vancoresmycin and its pharmaceutically acceptable salts and derivatives for use as pharmaceuticals and to the use of Vancoresmycin and its pharmaceutically acceptable salts and derivatives for the production of medicaments having antibacterial activity. The present invention further relates to pharmaceutical compositions which contain an effective amount of Vancoresmycin and/or one or more pharmaceutically acceptable salts and/or derivatives thereof, together with a pharmaceutically acceptable carrier.
Vancoresmycin can be administered orally, intramuscularly, intravenously, or by other modes of administration. Pharmaceutical compositions which contain Vancoresmycin or a pharmaceutically acceptable salt or derivative thereof with other pharmaceutically active substances can be prepared by mixing the active compounds with one or more pharmacologically tolerated auxiliaries and/or excipients such as, for example, fillers, emulsifiers, lubricants, masking flavors, colorants, or buffer substances, and converting the mixture into a suitable pharmaceutical form such as, for example, tablets, coated tablets, capsules, granules, powders, emulsions, suspensions, or solutions suitable for parenteral administration.
Examples of auxiliaries and/or excipients which may be mentioned are tragacanth, lactose, talc, agarxe2x80x94agar, polyglycols, ethanol, and water. Suitable and preferred for parenteral administration are suspensions or solutions in water. It is also possible to administer the active substances as such, without vehicles or diluents, in a suitable form, for example, in capsules.
As is customary, the galenic formulation and the method of administration as well as the dosage range which are suitable in a specific case depend on the species to be treated and on the state of the respective condition or disease, and can be optimized using methods known in the art. On average, the daily dose of active compound in a patient of about 75 kg weight is at least 0.001 mg to at most 10 mg, typically at most 10 mg.